1. Field of the Invention
The present invention relates to pharmaceutical compositions containing a alkylaryl polyether alcohol polymer. More specifically, the present invention relates to pharmaceutical compositions containing alkylaryl polyether alcohol polymer tyloxapol and to methods for treating respiratory inflammation with the pharmaceutical compositions.
2. The Prior Art
Discussion Of Oxidant-Mediated Injury
Oxygen is life-giving to aerobic plants and animals who depend on it for energy metabolism. It can also be lethal to those same organisms when it is altered from its stable dioxygen (O.sub.2) state to any one of three partially reduced species: a) the one electron reduced form superoxide anion (O.sub.2.sup.-); b) the two electron reduced form hydrogen peroxide (H.sub.2 O.sub.2); or the deadly three electron reduced form the hydroxyl radical (.sup..cndot. OH). In biologic systems O.sub.2.sup.- and H.sub.2 O.sub.2 are metabolic byproducts of a host of enzymes (oxygenases) that use oxygen as a cofactor. H.sub.2 O.sub.2 is also produced from O.sub.2.sup.- by the enzymatic action of superoxide dismutases. However, .sup..cndot. OH is generally produced only when O.sub.2.sup.- and H.sub.2 O.sub.2 interact with transitional ions of metals such as iron and copper in dangerous cyclical redox reactions: EQU Fe.sup.3+ +.sup..cndot. O.sub.2.sup.- .fwdarw..fwdarw..fwdarw.Fe.sup.2+ +O.sub.2 EQU Fe.sup.2+ +H.sub.2 O.sub.2 .fwdarw..fwdarw..fwdarw.Fe.sup.3+ +.sup..cndot. OH+-OH
The above reactions are termed the superoxide-driven Fenton reaction common in biological systems. The Fenton reaction can also be initiated by other reducing substances such as ascorbate in the presence of ferric iron and H.sub.2 O.sub.2.
While .sup..cndot. O.sub.2.sup.- and H.sub.2 O.sub.2 are each toxic for biological systems, .sup..cndot. OH (and its alternate hypothesized form the ferryl intermediate FeO.sup.2+) is a highly reactive species that can oxidize unsaturated membrane lipids, damage cellular proteins and cause mutagenic strand breaks in DNA. To prevent injury from partially reduced O.sub.2 species under normal conditions, cells have evolved an elaborate system of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and antioxidant molecules (glutathione, alpha-tocopherol, beta carotene). However, when production of partially reduced O.sub.2 species exceeds the capacity of cellular antioxidant defenses to contain them, oxidant injury occurs.
A growing number of mammalian disease entities are now thought to be related to overproduction of partially reduced O.sub.2 species, including the reperfusion injury syndromes myocardial infarction and stroke, adult respiratory distress syndrome, oxygen toxicity of the lung, lung injury from asbestos, Parkinson's disease, thermal and solar burns of the skin, and injury to the gastrointestinal tract from nonsteroidal anti-inflammatory agents (see Table IV, page 60, Halliwell B and Gutteridge J M C. Methods in Enzymology (1990) 186:1-85). Treatment of these conditions is increasingly directed either toward strategies that prevent enzymatic production of partially reduced O.sub.2 species and to the introduction of exogenous antioxidant compounds that restore oxidant-antioxidant balance in biologic and chemical systems. More recently, as will be outlined below, treatment of inflammation in many of these conditions has been directed toward interrupting activation of the transcription factors mediating the genetic expression of pro-inflammatory cytokines important in the pathogenesis of these conditions.
Discussion Of Transcription Factors And Cytokines
Transcription factors are cellular proteins that bind to regulatory sequences of genes and increase or decrease the rate of gene transcription. By affecting the rate of gene transcription, transcription factors play a critical role in regulation of cell function during health and disease. Among the most important transcription factors in disease are those that regulate expression of the genes for pro-inflammatory cytokines. These cytokines are secreted cellular proteins that dramatically affect the behavior of other cells. As examples, the cytokine TNF-.alpha. causes weight loss in patients with tumors or chronic infections, produces cellular death and is thought to be an important mediator of septic shock. The cytokine IL-1.beta. mediates fever, and shares many of the properties of TNF. The cytokine IL-8 (and its close relatives such as RANTES) is a potent chemotactic signal aiding in the recruitment of inflammatory cells such as neutrophils. GM-CSF signals the bone marrow to produce more inflammatory cells, activates those cells once produced and lengthens their survival. These cytokines play important roles in mediating the pathogenesis of such inflammatory diseases as cystic fibrosis, chronic bronchitis, asthma and viral infections, among many others (T. L. Bonfield, et al. "Inflammatory cytokines in cystic fibrosis lungs". American Journal of Respiratory and Critical Care Medicine (1996) In Press; N. G. McElvaney, et al. "Modulation of airway inflammation in cystic fibrosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor". Journal of Clinical Investigation (1992) 90:1296-1301; K. D. Pfeffer, et al. "Expression and regulation of tumor necrosis factor in macrophages from cystic fibrosis patients". American Journal of Respiratory, Cell and Molecular Biology (1993) 9:511-519; G. Williams and B. P. Giroir. "Regulation of cytokine gene expression: Tumor necrosis factor, interleukin-1, and the emerging biology of cytokine receptors". New Horizons (1995) 3:276-287; C. A. Dinarello. "Role of interleukin-1 and tumor necrosis factor in systemic responses to infection and inflammation". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 211-232; W. C. Greene. "The interleukins". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I. Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 233-245; M. Baggiolini, et al. "Interleukin-8 and related chemotactic cytokines". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I. Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 247-263; D. W. Golde and G. C. Baldwin. "Myeloid growth factors". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I. Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 291-301; R. J. Horwitz and W. W. Busse. "Inflammation and asthma". Clinics in Chest Medicine (1995) 16:583-602).
These cytokines share regulation of their expression by the transcription factor Nuclear Factor kappa-B (NF-.kappa.B), a particularly important transcription factor mediating inflammatory events (U. Siebenlist, G. Granzuso and R. Brown. "Structure, regulation and function of NF-.kappa.B". Annual Review of Cell Biology (1994) 10:405-455). NF-.kappa.B is also an important transcriptional regulator chemokines such as RANTES (U. Siebenlist, G. Granzuso and R. Brown. "Structure, regulation and function of NF-.kappa.B". Annual Review of Cell Biology (1994) 10:405-455) and of inducible nitric oxide synthase (iNOS) (P. J. Nelson, et al. "Genomic organisation and transcriptional regulation of the RANTES chemokine gene". Journal of Immunology (1993) 151:2601-2612), the enzyme producing nitric oxide (NO), a critical oxidant chemical produced as part of the pathogenesis of septic shock. NF-.kappa.B is present in the cytoplasm in an inactive form complexed to an inhibitory protein I.kappa.B. A number of events, yet to be completely characterized, cause I.kappa.B to dissociate from NF-.kappa.B in the cytoplasm. Free NF-.kappa.B then localizes to the nucleus, where it binds to a specific .kappa.B recognition site in the promoter region of target genes, prompting their expression. NF-.kappa.B is activated by a number of stimuli, including cytokines themselves, and by lipopolysaccharide (LPS) (U. Siebenlist, G. Granzuso and R. Brown. "Structure, regulation and function of NF-.kappa.B". Annual Review of Cell Biology (1994) 10:405-455). NF-.kappa.B is also activated by oxidants such as hydrogen peroxide (M. Meyer, R. Schreck, and P. A. Baeverie. "H.sub.2 O.sub.2 and antioxidants have opposite effects on the activation of NF-.kappa.B and AP-1 in intact cells: AP-1 as secondary antioxidant response factor". EMBO Journal (1993) 12:2005-2015), suggesting that it may be an oxidant-stress responsive transcription factor. Conversely, some of the most potent inhibitors of NF-.kappa.B activation are compounds which can also act as antioxidants. A few, but not most, antioxidants prevent activation of NF-.kappa.B by LPS, prevent increases in corresponding messenger RNAs for inflammatory cytokines and decrease levels of TNF and IL-1 in the circulation following LPS injection (E. M. Eugui, et al. "Some antioxidants inhibit, in a coordinate fashion, the production of tumor necrosis factor .alpha., IL-1.beta. and IL-6 by human peripheral blood mononuclear cells". International Journal of Immunology (1993) 6:409-422; R. Schreck, et al. "Dithiocarbamates as potent inhibitors of nuclear factor .kappa.B activation in intact cells". Journal of Experimental Medicine (1992) 175:1181-1194). However, the few antioxidants known to inhibit NF-.kappa.B activation share no common structural similarity distinguishing them from those antioxidants that fail to prevent activation of NF-.kappa.B (see Eugui, above), preventing one skilled in the art from predicting which antioxidant compounds will and which will not favorably reduce NF-.kappa.B activation as a strategy of ameliorating inflammatory events in disease.
Another class of compounds known to inhibit NF-.kappa.B activation are anti-inflammatory corticosteroids. Corticosteroids act by combining in the cytoplasm with an intracellular protein called the Glucocorticoid Receptor (GR). Previously, the anti-inflammatory action of corticosteroids was thought to occur exclusively as a result of passage of the GR-steroid complex to the nucleus, where the complex attaches to and influences regulatory gene regions called Glucocorticoid Responsive Elements (GREs). However, recently it has been shown that a major mechanism of anti-inflammatory glucocorticoid activity is inhibition of NF-.kappa.B (I. M. Adcock, et al. "Effects of glucocorticoids on transcription factor activation in human peripheral blood mononuclear cells". American Journal of Physiology (1995) 268(Cell Physiology 37):C331-C338). The GR-steroid complex prevents activation of NF-.kappa.B by directly interacting with free NF-.kappa.B in the cytoplasm, preventing NF-.kappa.B from translocating to the nucleus (A. Ray and K. E. Prefontaine. "Physical association and functional antagonism between the p65 subunit of transcription factor NF-.kappa.B and the glucocorticoid receptor". Proceedings of the National Academy of Sciences, USA (1994) 91:752-756). However, the GR-steroid complex accomplishes inhibition of NF-.kappa.B by mutual repression. By combining with free NF-.kappa.B in the cytoplasm, it too is kept from translocating to the nucleus to up-regulate other anti-inflammatory events. Indeed, mutual repression is thought to explain in part the phenomenon of steroid resistance in severe asthmatics. IL-1, IL-6, TNF and other pro-inflammatory cytokines secreted in the airway during an asthma attack increase cellular activation of NF-.kappa.B, providing more NF-.kappa.B subunits to bind GR-steroid complexes, reducing the amount of GR-steroid complex available to translocate to the nucleus (P. J. Barnes, A. P. Greening and G. K. Crompton. "Glucocorticoid resistance in asthma". American Journal of Respiratory and Critical Care Medicine (1995) 152:S125-S142).
Discussion Of Alkylaryl Polyether Alcohol Polymers, Including Tyloxapol
Antioxidants are compounds that can be easily oxidized to stable chemical forms. They can protect chemical and biologic systems by sacrificing themselves to oxidation in preference to oxidation of critically important chemical and biological molecules. Not all oxidizable compounds can perform antioxidant function. To successfully protect chemical and biologic systems from oxidants, the antioxidant must have a higher reactivity for the oxidant than the chemical or biologic molecule which it seeks to protect. To protect the desired chemical and biologic system from oxidation, it is also necessary for the antioxidant to partition itself adjacent to the molecule to be protected. As an example, a molecule to be protected within the lipid bilayer of plasma, endosomal or nuclear membranes might be best protected by an antioxidant with, at least in part, a lipophilic structure, so that it is partitioned to or near the lipid portion of the membrane, adjacent to the molecule needing protection from oxidation.
It has recently been shown that a previously known class of drugs, the alkylaryl polyether alcohol polymers, are potent antioxidants useful in the treatment of mammalian diseases (U.S. Pat. No. 5,474,760 issued 1995 to Ghio, Kennedy and Piantadosi, assignors to Duke University, and U.S. Ser. No. 08/039/732). Alkylaryl polyether alcohol polymers are used commercially as surface active detergents and wetting agents (U.S. Pat. No. 2,454,541, issued in 1948 to Bock and Rainey, assignors to Rohm & Haas). The best known of this class is tyloxapol, a polymer of 4-(1,1,3,3-tetramethylbutyl)phenol with formaldehyde and oxirane. However, other compounds in the class, sharing the properties of tyloxapol, are well known in the art (J. W. Cornforth, et al. "Antituberculous effect of certain surface-active polyoxyethylene ethers in mice". Nature (1951) 168:150-153).
On alkylaryl polyether alcohol polymer used previously in aerosol pharmacologic formulations is tyloxapol, or Triton WR-1339 (M. L. Tainter, et al. "Alevaire as a mucolytic agent". New England Journal of Medicine (1955) 253:764-767). A composition sold by Winthrop Laboratories (a division of Sterling Drug, Inc.) and by Breon Laboratories (subsidiary of Sterling Drug, Inc.) under the trademark ALEVAIRE.RTM., containing 0.125% aqueous SUPERINONE.RTM. (brand of tyloxapol) in combination with 2% sodium bicarbonate and 5% glycerin, had been marketed for about 30 years for treatment of mucous secretions in patients with diseases and disorders such as chronic bronchitis, croup, pertussis, and poliomyelitis. (See, for example, a product brochure entitled "ALEVAIRE.RTM. Detergent Aerosol for Inhalation" (November, 1961) distributed by Breon Laboratories.).
At the time the ALEVAIRE formulation new drug application (NDA) was approved in the early 1950's, the Federal Food, Drug, and Cosmetic Act (FDA Act) did not require FDA to consider efficacy in the drug approval process. In 1962, the FDA Act was amended to require FDA to consider efficacy, and to authorize the agency to remove from the market drugs with approved NDAs if substantial evidence was lacking that the drug was ineffective for its intended use. To comply with the latter legislative mandate, FDA established the Drug Efficacy Study Implementation (DESI) review. ALEVAIRE was considered in the DESI review, and was found to be ineffective. In July of 1968, FDA notified its sponsor, Sterling Drug. Sterling appealed the FDA's findings (Sterling Drug, Inc., v. Weinberger, 503F.2d 675 (2d Cir. 1974), 384 F. Supp. 675 (S.D.N.Y. 1974), and 509 F.2d 1236 (2d Cir. 1975)). The legal battle lasted 13 years; it was not until 1981, after a formal evidentiary public hearing, that FDA published an adverse "final decision" on ALEVAIRE that was not appealed by Sterling (ALEVAIRE; Final Decision Following Formal Evidentiary Public Hearing in Adjudicatory Proceeding, 46 Fed. Reg 56043 (Nov. 13, 1981)).
FDA found that there was no evidence that the tyloxapol in ALEVAIRE.RTM. had any effect on secretions in the lung from diseases such as chronic bronchitis other than that of water in thinning secretions by simple dilution, and that papers in the manufacturer's bibliography were based on clinical impression and did not reflect adequate controls. (See, letter dated May 27, 1994 to Dr. Thomas Kennedy, one of the co-inventors of the present application, from Ms. Carolann W. Hooton, Chief, Freedom of Information Office, Center for Drug Evaluation and Research, Department of Health & Human Services, Public Health Service, Food and Drug Administration, Rockville, Md.). Surprisingly, the present inventors have found that alkylaryl polyether alcohol polymers of the class typified by tyloxapol, are potent antioxidants, inhibitors of the activation of NF-.kappa.B (see Example IV below) and inhibitors of cellular production of pro-inflammatory cytokines (see Example V below).
Even before its withdrawal from the market, there was published evidence that the ALEVAIRE formulation of tyloxapol was associated with side effects in some individuals. Paez and Miller studied ALEVAIRE in 20 patients with chronic obstructive pulmonary disease (Paez, P. N. and W. F. Miller. 1971. Surface active agents in sputum evacuation: a blind comparison with normal saline solution and distilled water. Chest 60:312-317). Lung function did not change after subjects inhaled solutions of normal saline, water, or Tergemist (sodium 2-ethylehexyl sulfate 0.125% and potassium iodide 0.1%), but four patients developed evidence of increased airways obstruction after inhaling ALEVAIRE. Subsequently, Fevrier and Bachofen, using a double-blind crossover design, studied the effect of ALEVAIRE or saline as carrier solutions for the inhalation of beta agonists in 24 patients with asthma (Fevrier, D., and H. Bachofen. 1975. Vergleich von tyloxapol (Tacholiquin, ALEVAIRE) mit physiologischer kochsalzlosung als inhalationstragerluscungen. Schweiz. med Wschr. 195:810-815). The authors measured specific airway conductance (the inverse of airways resistance) over a 2 hour period following inhalation of 3 ml of test solution. ALEVAIRE solution without beta agonist bronchodilator caused a 20% fall in specific conductance at 20 minutes (p&lt;0.05) that resolved completely by 60 minutes. Thus, the ALEVAIRE formulation was clearly causes bronchospasm after inhalation by susceptible individuals such as those with asthma or airways reactivity.
The present aerosol formulation containing tyloxapol is EXOSURF.RTM. NEONATAL, approved by the FDA in 1990 and marketed by Glaxo Welcome as an intratracheally instilled suspension for the treatment of neonatal respiratory distress syndrome. EXOSURF is a formulation of 108 mg diphalmitoylphosphatidyl choine (DPPC), 12 mg cetyl alcohol, 8 mg tyloxapol and 47 mg sodium chloride, reconstituted with 8 ml sterile water. DPPC is thought to be the major functional component. Tyloxapol is added as a dispersing agent so that DPPC can remain an emulsion when reconstituted. When reconstituted, the EXOSURF solution contains 13.5 mg/ml DPPC, 1.5 mg/ml cetyl alcohol, and 1 mg/ml tyloxapol in 0.1 N NaCl. The product is used for both prophylactic and rescue treatment of infants. Neonates treated prophylactically are recommended to receive 3 doses of 5 ml/kg at 12 hour intervals after birth. A number of major adverse effects are seen occasionally seen after EXOSURF administration, including reflux of EXOSURF into the endotracheal tube after intratracheal administration, mucus plugging shortly after administration, pulmonary hemorrhage in low birth weight infants, and arterial oxygen desaturation (EXOSURF Neonatal. 1995. Physicians Desk Reference. Medical Economics, Montvale, N.J. 758-762). EXOSURF has also undergone a trial for sepsis-induced adult respiratory distress syndrome in adults (Weg, J. G., R. A. Balk, et al. 1994. Safety and potential efficacy of an aerosollized surfactnat in human sepsis-induced adult respiratory distress syndrome. J.A.M.A. 727:1433-1438). Subjects received EXOSURF aerosolized continuously over 12 or 24 hours, respectively for up to 5 days (568.4.+-.53.6 grams in the 12 hour group and 1,128.4.+-.99.3 grams in the 24 hour EXOSURF group). Because of the lipid DPPC component, the aerosol emulsion formulation of EXOSURF tended to accumulate and occlude the exhalation bacterial filter on the mechanical ventilator. One subject suffered a pneumothorax (ruptured lung) as a consequence of this occlusion, when pressure in the ventilator circuit built up and could not escape due to an exhalation valve blocked by the accumulated lipid emulsion.
Synopsis Of Background Discussion
Inflammation in a multitude of diseases is mediated by activation of the transcription factor NF-.kappa.B, which in turn causes an increase in cellular production of pro-inflammatory cytokines such as TNF, IL-1, IL-6, IL-8 and the growth factor GM-CSF, and an increase in critical cellular enzymes, such as inducible nitric oxide synthase (iNOS). The current treatment available to prevent activation of NF-.kappa.B and subsequent cytokine secretion is anti-inflammatory glucocorticoids. Recently a few, but not most, antioxidants have been found to also inhibit NF-.kappa.B.
It is theoretically possible to synthesize a multitude of compounds with antioxidant properties. However, there is no predictable structural similarity among the few agents shown to prevent NF-.kappa.B activation. Thus, the demonstration that a compound shows antioxidant activity would not, in of itself, predict that the same compound would also inhibit NF-.kappa.B activation and secretion of pro-inflammatory cytokines. Also, the factor limiting use of antioxidants as treatments in biologic systems is the inherent toxicity of many antioxidant compound themselves. Likewise, anti-inflammatory cortosteroids are potent inhibitors of NF-.kappa.B, but their use as such is severely limited by the well-known side effects of corticosteroids, including glucose intolerance, hypertension, bone resorption, weight gain and cataracts. Thus, it is a major advantage to discover that a class of commonly used and nontoxic ingredients in medicinal pharmacologic preparations are not only potent antioxidants, but also potent inhibitors of NF-.kappa.B activation. Not only can such compounds be used as treatments for diseases where antioxidants might be predicted to be of value, but they can be used as treatments for NF-.kappa.B mediated inflammatory conditions without themselves causing toxicity to biologic systems.
The findings presented in the various examples to follow will demonstrate that tyloxapol is a potent antioxidant that also prevents NF-.kappa.B activation and suppresses secretion of inflammatory cytokines. These features of tyloxapol would make it a useful anti-inflammatory drug treatment strategy for various mammalian diseases, especially diseases of the respiratory tract. However, the current formulations of tyloxapol, ALEVAIRE and EXOSURF, have undesirable features, such as increasing airways resistance in asthmatics, in the case of ALEVAIRE, or production of plugging of airways and ventilator circuits, in the case of EXOSURF.